Return to study ST001524 main page
MB Sample ID: SA128430
| Local Sample ID: | Cell_9312_A |
| Subject ID: | SU001598 |
| Subject Type: | Other |
| Subject Species: | Prochlorococcus marinus str. MIT 9312;Prochlorococcus marinus MIT9313 |
| Taxonomy ID: | 74546;74547 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001598 |
| Subject Type: | Other |
| Subject Species: | Prochlorococcus marinus str. MIT 9312;Prochlorococcus marinus MIT9313 |
| Taxonomy ID: | 74546;74547 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| Cell_9312_A | SA128430 | FL016027 | Pellet | Sample Type |
| Cell_9312_A | SA128430 | FL016027 | 9312 | Cell Type |
Collection:
| Collection ID: | CO001593 |
| Collection Summary: | Axenic cultures of Prochlorococcus strain MIT9312 and MIT9313 were grown in defined artificial AMP1 media supplemented with 10 mM (final concentration) filter-sterilized sodium bicarbonate. Seven 20 L cultures were grown for each of the two Prochlorococcus strains, providing three replicates for the lipid and small metabolite analysis and an additional sample for proteomics analysis. Strains MIT9312 and MIT9313 are available from the National Center for Marine Algae and Microbiota. 20 L cultures were grown in polycarbonate carboys (ThermoFisher Nalgene, Waltham, MA, USA) with gentle stirring (60 rpm), under constant light flux (10 – 20 µmol Q m -2 s -1 for MIT9313; 30 – 40 µmol Q m -2 s -1 for MIT9312) at 24°C. Vesicles were collected from 20 L cultures of Prochlorococcus during mid-to-late exponential growth phase and isolated as described previously (Biller et al., 2014, Science). Briefly, cultures were first gravity filtered through a 0.2 µm capsule filter (Polycap 150TC; GE Life Sciences/Whatman, Maidstone, UK). The filtrate was then concentrated using a 100 kDa tangential flow filter (Ultrasette with Omega membrane; Pall, Port Washington, NY, USA) and re-filtered through a 0.2 µm syringe filter. Vesicles were pelleted from the sample by ultracentrifugation at ~100,000 x g (Beckman-coulter SW32Ti rotor; 32,000 rpm, 1.5 hrs, 4°C), purified on an OptiPrep gradient (Biller et al., 2014, Science), then washed and resuspended in 0.2 µm filtered 1x PBS. |
| Sample Type: | Cultured Prochlorococcus cells and vesicles |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR001613 |
| Treatment Summary: | No treatment - cells and vesicles were cultured according to standard protocols. We used targeted and untargeted metabolomics to characterize the metabolome of cells and vesicles. |
Sample Preparation:
| Sampleprep ID: | SP001606 |
| Sampleprep Summary: | Each sample was extracted using a modified Bligh-Dyer extraction. Briefly, quantitative aliquots of cell pellets were transferred into 15 mL teflon centrifuge tubes containing a mixture of 100 µm and 400 µm silica beads. Quantitative aliquots of extracellular vesicles were transferred into 24 mL glass vials and extracted without bead beating. Heavy isotope-labeled internal standards were added along with ~2 mL of cold aqueous solvent (50:50 methanol:water) and ~3 mL of cold organic solvent (dichloromethane). The samples were shaken on a FastPrep-24 Homogenizer for 30 seconds and chilled in a -20 °C freezer repeatedly for three cycles of bead-beating and a total of 30 minutes of chilling. The organic and aqueous layers were separated by spinning samples in a centrifuge at 4,300 rpm for 2 minutes at 4 °C. The aqueous layer was removed to a new glass centrifuge tube. The remaining organic fraction was rinsed three more times with additions of 1 to 2 mL of 50:50 methanol:water. All aqueous rinses were combined for each sample and ~2 mL of cold dichloromethane was added to the combined aqueous layer. Tubes were shaken and centrifuged at 4,300 rpm for 2 minutes at 4°C. The aqueous layer was removed to a new glass vial and dried under N2 gas. The remaining organic layer in the bead beating tubes was transferred into the glass centrifuge tube and the bead beating tube was rinsed two more times with cold organic solvent. The combined organic rinses were centrifuged, transferred to a new glass vial, and dried under N2 gas. Dried aqueous fractions were re-dissolved in 380 µL of water. Dried organic fractions were re-dissolved in 380 µL of 1:1 water:acetonitrile. 20 µL of isotope-labeled injection standards in water were added to both fractions. Process blanks (MilliQ water), media blanks, and PBS (vesicle suspension buffer) were extracted and analyzed alongside each sample set. |
| Processing Storage Conditions: | On ice |
| Extraction Method: | Bligh-Dyer |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN002542 | AN002543 | AN002544 | AN002545 |
|---|---|---|---|---|
| Analysis type | MS | MS | MS | MS |
| Chromatography type | HILIC | HILIC | Reversed phase | Reversed phase |
| Chromatography system | Waters Acquity I-Class | Waters Acquity I-Class | Waters Acquity I-Class | Waters Acquity I-Class |
| Column | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) | Waters Acquity UPLC HSS Cyano (100 x 2.1mm,1.8um) | Waters Acquity UPLC HSS Cyano (100 x 2.1mm,1.8um) |
| MS Type | ESI | ESI | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap | Thermo Q Exactive HF hybrid Orbitrap | Thermo Q Exactive HF hybrid Orbitrap | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE | POSITIVE | POSITIVE |
| Units | Adjusted and normalized peak areas | Adjusted and normalized peak areas | Adjusted and normalized peak areas | Adjusted and normalized peak areas |
Chromatography:
| Chromatography ID: | CH001862 |
| Chromatography Summary: | See attached summary. |
| Methods Filename: | Ingalls_Lab_LC_Methods.txt |
| Instrument Name: | Waters Acquity I-Class |
| Column Name: | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| Column Temperature: | 30 |
| Flow Rate: | 0.15 ml/min |
| Solvent A: | 85% acetonitrile/15% water; 10 mM ammonium carbonate |
| Solvent B: | 15% acetonitrile/85% water; 10 mM ammonium carbonate |
| Chromatography Type: | HILIC |
| Chromatography ID: | CH001863 |
| Chromatography Summary: | See attached summary |
| Methods Filename: | Ingalls_Lab_LC_Methods.txt |
| Instrument Name: | Waters Acquity I-Class |
| Column Name: | Waters Acquity UPLC HSS Cyano (100 x 2.1mm,1.8um) |
| Column Temperature: | 35 |
| Flow Rate: | 0.4 ml/min |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% acetonitrile; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS002360 |
| Analysis ID: | AN002542 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | See attached protocol. |
| Ion Mode: | POSITIVE |
| Analysis Protocol File: | Ingalls_Lab_MS_Methods.txt |
| MS ID: | MS002361 |
| Analysis ID: | AN002543 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | See attached protocol. |
| Ion Mode: | NEGATIVE |
| Analysis Protocol File: | Ingalls_Lab_MS_Methods.txt |
| MS ID: | MS002362 |
| Analysis ID: | AN002544 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | See attached protocol. Data from aqueous fraction. |
| Ion Mode: | POSITIVE |
| Analysis Protocol File: | Ingalls_Lab_MS_Methods.txt |
| MS ID: | MS002363 |
| Analysis ID: | AN002545 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | See attached protocol. Data from organic fraction. |
| Ion Mode: | POSITIVE |
| Analysis Protocol File: | Ingalls_Lab_MS_Methods.txt |
